Temporal response of canine flexor tendon to limb suspension

Tendon disuse, or stress deprivation, frequently accompanies clinical disorders and treatments, yet the metabolism of tendons subject to stress deprivation has rarely been investigated systematically. The effects of stress deprivation on canine flexor tendon were investigated in this study. One adult canine forepaw was suspended for 21 or 42 days. Control forepaws were collected from dogs that had no intervention on their limbs and paws. The expression of collagen I and III was not significantly altered in the tendons disused for 21 days but was significantly decreased at 42 days (P < 0.03). The expression of collagen II, aggrecan, decorin, and fibronectin was significantly decreased in the tendons in the suspended limbs at 21 days (P < 0.002) and further reduced at 42 days. With stress deprivation, the expression of matrix metalloproteinase 2 (MMP2) was significantly increased (P < 0.004) at 21 and 42 days. The expression of MMP3 was significantly decreased at 21 and 42 days (P < 0.03). The expression of MMP13 was not altered with stress deprivation at 21 and 42 days. The expression of MMP14 was significantly increased at 21 days (P = 0.0015) and returned to the control level at 42 days. Tissue inhibitor of metalloproteinase 1 (TIMP1) expression was decreased after the limbs were suspended for 42 days (P = 0.0043), but not 21 days. However, TIMP2 expression was not significantly different from control at 21 or 42 days. Furthermore, the cross-sectional area of the stress-deprived tendons at 42 days was decreased compared with the control group (P < 0.01). The intervention method in this study did not result in any alteration of stiffness of the tendon. Our study demonstrated that stress deprivation decreases the anabolic process and increases the catabolic process of extracellular matrix in flexor tendon.     

Prenatal diagnosis of Dandy-Walker malformation associated with distal limb deficiencies

We report the perinatal findings of a 23 gestational-week fetus with Dandy-Walker malformation (DWM), ventriculomegaly, symmetrical transverse limb deficiencies, hypertelorism, frontal bossing, low-set ears, and a depressed nasal bridge. The karyotype was 46,XX. We believe that this combination is significant. Concomitant DWM and symmetrical distal limb deficiencies may represent a new entity that awaits more new cases for further delineation.     

Microlymphaticovenous anastomosis in the treatment of lower limb obstructive lymphedema: analysis of 91 cases

Since 1980, 110 cases of lymphedema have been treated by microlymphaticovenous anastomosis. Of these 110 patients, 91 with obstructive lymphedema of lower limbs were reviewed. The immediate and long-term results have been very satisfactory. Excellent and good results were obtained in 79.1 percent. An average reduction in circumference of the affected limb of 6.4 cm and an average reduction of excess volume of 59.2 +/- 29.5 percent (representing 703 +/- 850 ml) were obtained. Subjective symptoms and objective signs were improved. Four patients (4.4 percent) showed poor results owing to severe fibrosis of neighboring tissue; no lymphatics could be located for anastomosis. As the authors gained experience with the operation over the last 3 years, they modified the operative procedure, the anastomotic technique, and the selection of collective lymphatics. The data obtained suggest that the quality of results is proportional to the number of anastomoses. In order to obtain the best results, the criteria for selection of patients and avoidance of postoperative relapse are discussed. Finally, a test for determination of the indications for microlymphatic surgery is described.     

A model to predict canine pelvic limb musculoskeletal geometry

We developed a model to predict the three-dimensional canine pelvic limb muscular geometry (i.e., all muscle moment arms during any instant in gait). Forty-one muscle origins and insertions, as well as external landmarks (to obtain anthropometric dimensions) were marked on both pelvic limbs of five dogs and digitized on biplanar radiographs. Reference frames in the pelvis, femur, and tibia established the three-dimensional coordinates of each origin, insertion, and landmark. A set of dimensionless 'scaled coordinates' was created by dividing the actual origin and insertion coordinates by selected anthropometric dimensions of each animal. Combining scaled coordinates from all ten limbs produced an averaged 'template' of scaled coordinates. To provide limited validation of the scaling procedure, we measured the anthropometric dimensions between externally palpable landmarks of two additional pelvic limbs. The anthropometric dimensions were multiplied by the averaged template coordinates to calculate two new sets of hindlimb muscle coordinates within the three bony reference frames. The two limbs then were dissected, muscle endpoints were marked, and biplanar radiographs of each of the limb segments were digitized. The actual coordinates so obtained were similar to those predicted by the template and anthropometric measures.     

Wls-mediated Wnts differentially regulate distal limb patterning and tissue morphogenesis

Wnt proteins are diffusible morphogens that play multiple roles during vertebrate limb development. However, the complexity of Wnt signaling cascades and their overlapping expression prevent us from dissecting their function in limb patterning and tissue morphogenesis. Depletion of the Wntless (Wls) gene, which is required for the secretion of various Wnts, makes it possible to genetically dissect the overall effect of Wnts in limb development. In this study, the Wls gene was conditionally depleted in limb mesenchyme and ectoderm. The loss of mesenchymal Wls prevented the differentiation of distal mesenchyme and arrested limb outgrowth, most likely by affecting Wnt5a function. Meanwhile, the deletion of ectodermal Wls resulted in agenesis of distal limb tissue and premature regression of the distal mesenchyme. These observations suggested that Wnts from the two germ layers differentially regulate the pool of undifferentiated distal limb mesenchyme cells. Cellular behavior analysis revealed that ectodermal Wnts sustain mesenchymal cell proliferation and survival in a manner distinct from Fgf. Ectodermal Wnts were also shown for the first time to be essential for distal tendon/ligament induction, myoblast migration and dermis formation in the limb. These findings provide a comprehensive view of the role of Wnts in limb patterning and tissue morphogenesis.     

Relative variation in human proximal and distal limb segment lengths

The pattern of variation and covariation of proximal and distal limb segment lengths was examined within and between 20 geographically diverse skeletal samples of modern humans. Analyses of variance-covariance matrices (VCMs) of logarithmically transformed (ln) variates of humerus, radius, femur, and tibia length were performed to test the following hypotheses: first, within populations, the distal and proximal segments will have equal relative (i.e., size-independent) variability. However, between populations, the tibia is predicted to be more variable than the other segments. Tests of fit of computed VCMs to theoretical matrices by an iterative procedure (Anderson [1973] Ann. Stat. 1:135-141) reject the equal variance hypotheses, rather suggesting that the relative variances of the distal limb segments are greater than are those of the proximal. Males and females differ somewhat in that within females, the distal segments of both limbs have equal variance, while within males, the tibia has greater relative variance than the radius. The second hypothesis, regarding between-group variability, is somewhat supported in that between human populations, one cannot reject that the tibia has greater relative variance than the other limb segments. However, neither can one reject an alternative hypothesis that both distal limb segments (tibia and radius) are more variable than the proximal segments. Differential growth allometry is explored, and likely plays a major role in differences seen both within and between human populations.     

Stimulus-dependent pacemaker activity in the distal canine lower esophageal sphincter

Electrical and mechanical properties of the distal canine lower esophageal sphincter were studied in vitro to investigate possible means of inducing pacemaker activity. Both direct excitation and block of potassium conductance were investigated. The acetylcholine analog, carbachol, induced tissue depolarization and increase in tone but no electrical slow waves. Tetraethylammonium (TEA) chloride induced depolarization and evoked continuous spiking activity and increase in tone. BaCl did not depolarize the tissue but low amplitude spiking activity developed and increased tone. The putative potassium channel blocker, aminacrine at 2 X 10(-4) M, induced electrical slow wave activity in the distal lower esophageal sphincter, with or without superimposed spikes, accompanied by phasic contractile activity. This activity closely resembled the spontaneous pacemaker activity observed previously in the proximal lower esophageal sphincter. The aminacrine-induced activity was abolished by calcium influx blockers. Aminacrine, but not TEA or BaCl, abolished the nonadrenergic nerve-mediated inhibitory junction potentials. In conclusion, block of inhibitory innervation, and induction of electrical slow waves as a control mechanism for phasic contractile activity, seems to require blockade of an aminacrine- but not TEA-sensitive potassium conductance.     

Bowl is required downstream of Notch for elaboration of distal limb patterning

In the Drosophila leg, activation of Notch leads to the establishment of the joints that subdivide the appendage into segments. We find that mutations in bowl result in similar phenotypes to Notch, causing fusion and truncations of tarsal segments (tarsomeres) and, like its close relative Odd-skipped, Bowl is produced in response to Notch signalling at a subset of segment boundaries. However, despite the fact that bowl mutant clones result in fusion of tarsomeres, Bowl protein is only found at the t1/tibial and t5/pretarsal boundaries, not at tarsomere joints. One hypothesis to reconcile these data is that bowl has a role at an earlier stage in tarsal development. We therefore investigated the effects of bowl mutations on the expression of leg 'gap' genes that confer regional identity on the developing leg. Several of these genes have altered expression in bowl mutant cells. For example, bric-a-brac2 is normally expressed in the central part of the tarsus domain but expands into distal and proximal regions in bowl clones. Conversely, ectopic bowl leads to a reduction in bric-a-brac2, with a concomitant expansion of proximal (t1) and distal (t5) tarsomere fates. The bowl gene is therefore required for the elaboration of pattern in the tarsus and its effects suggest a progressive model for the determination of P/D identities. This mechanism might be important in the diversification of arthropod limbs, because it explains how segmented tarsomeres could have arisen from an ancestral limb with an unsegmented tarsus.     

Some distal limb structures develop in mice lacking Sonic hedgehog signaling

Patterning of the limb is coordinated by the complex interplay of three signaling regions: the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the non-ridge limb ectoderm. Complex feedback loops exist between Shh in the ZPA, Bmps and their antagonists in the adjacent mesenchyme, Wnt7a in the dorsal ectoderm and Fgfs in the AER. In contrast to the previously reported complete absence of digits in Shh(-/-) mice, we show that one morphologically distinct digit, with a well-delineated nail and phalanges, forms in Shh(-/-) hindlimbs, while intermediate structures are severely truncated and fused. The presence of distal autopod elements is consistent with weak expression of Hoxd13 in Shh(-/-) hindlimbs. Shh(-/-) forelimbs in contrast have one distal cartilage element, a less-well differentiated nail and fused intermediate bones. Interestingly, Ihh is expressed at the tip of Shh mutant limbs and could account for formation of distal structures. In contrast to previous studies we also demonstrate that Shh signaling is required for maintenance of normal Fgf8 expression, since expression of Fgf8, unlike some other AER marker genes, is rapidly lost from anterior to posterior after E10.5, with only a small domain of Fgf8 expression remaining posteriorly. Furthermore, loss of expanded Fgf8 expression is paralleled by a collapse of the handplate. Our data show that development of most intermediate elements of the hindlimb skeleton are Shh-dependent, and that Shh signaling is required for anterior-posterior expansion of the AER in both limbs and for the subsequent branching of zeugopod and autopod elements. Finally, we show that Shh is also required for outgrowth of the limb ectoderm and thus for the formation of a distinct limb compartment.     

Postmastectomy lymphedema

Disturbing lymphedema of the related arm occurs in 20 to 30 per cent of patients after conventional mastectomy.Infection, obesity and radiation therapy are the most important contributing factors in the development of swollen arm. Continuous suction drainage of the wound and the use of antibiotics will reduce the incidence of infection. Lymphangiosarcoma is a rare fatal complication of postmastectomy lymphedema.     

The apical ectodermal ridge is a timer for generating distal limb progenitors

The apical ectodermal ridge (AER) is a transient embryonic structure essential for the induction, patterning and outgrowth of the vertebrate limb. However, the mechanism of AER function in limb skeletal patterning has remained unclear. In this study, we genetically ablated the AER by conditionally removing FGFR2 function and found that distal limb development failed in mutant mice. We showed that FGFR2 promotes survival of AER cells and interacts with Wnt/beta-catenin signaling during AER maintenance. Interestingly, cell proliferation and survival were not significantly reduced in the distal mesenchyme of mutant limb buds. We established Hoxa13 expression as an early marker of distal limb progenitors and discovered a dynamic morphogenetic process of distal limb development. We found that premature AER loss in mutant limb buds delayed generation of autopod progenitors, which in turn failed to reach a threshold number required to form a normal autopod. Taken together, we have uncovered a novel mechanism, whereby the AER regulates the number of autopod progenitors by determining the onset of their generation.     

Creation and characterization of an immortalized canine myoblast cell line: Myok9

Mammalian Genome - The availability of an in vitro canine cell line would reduce the need for dogs for primary in vitro cell culture and reduce overall cost in pre-clinical studies. An immortalized...